This is a 371 of International Application PCT/GB99/04308 filed Dec. 17, 1999.
This invention relates to compounds that inhibit farnesylation of mutant ras gene products through inhibition of the enzyme farnesyl-protein transferase (FPTase). The invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer, which are mediated through farnesylation of ras.
Cancer is believed to involve alteration in expression or function of genes controlling cell growth and differentiation. Whilst not wishing to be bound by theoretical considerations the following text sets out the scientific background to ras in cancer. Ras genes are frequently mutated in tumours. Ras genes encode guanosine triphosphate (GTP) binding proteins which are believed to be involved in signal transduction, proliferation and malignant transformation. H-, K- and N-ras genes have been identified as mutant forms of ras (Barbacid M, Ann. Rev. Biochem. 1987, 56: 779-827). Post translational modification of ras protein is required for biological activity. Farnesylation of ras catalysed by FPTase is believed to be an essential step in ras processing. It occurs by transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to a cysteine at the C-terminal tetrapeptide of ras in a structural motif called the CAAX box. After further post-translational modifications, including proteolytic cleavage at the cysteine residue of the CAAX box and methylation of the cysteine carboxyl, ras is able to attach to the cell membrane for relay of growth signals to the cell interior. In normal cells activated ras is believed to act in conjunction with growth factors to stimulate cell growth. In tumour cells it is believed that mutations in ras cause it to stimulate cell division even in the absence of growth factors (Travis J, Science 1993, 260: 1877-1878), possibly through being permanently in GTP activated form rather than cycled back to GDP inactivated form. Inhibition of farnesylation of mutant ras gene products will stop or reduce activation.
One class of known inhibitors of farnesyl transferase is based on farnesyl pyrophosphate analogues; see for example European patent application EP 534546 from Merck. Inhibitors of farnesyl transferase based on mimicry of the CAAX box have been reported. Reiss (1990) in Cell 62, 81-8 disclosed tetrapeptides such as CVIM (Cys-Val-Ile-Met). James (1993) in Science 260, 1937-1942 disclosed benzodiazepine based peptidomimetic compounds. Lerner (1995) in J. Biol. Chem. 270, 26802 and Eisai in International Patent Application WO 95/25086 disclosed further peptidomimetic compounds based on Cys as the first residue. Bristol-Myers Squibb in European Patent Application EP 696593 disclosed farnesyl transferase inhibitors having a 4-sulfanylpyrrolidine residue in the first position.
International Patent Application WO 97/17070 discloses a broad range of compounds which can contain an imidazole group and a methionine substituent. We have now discovered a narrow class of imidazole compounds which have improved properties.
According to one aspect of the present invention there is provided a compound of Formula (1): 
wherein Ar1 represents: 
R5 is hydrogen, C1-4alkyl, phenylC1-4alkyl;
R6 is hydrogen, C1-4alkyl, hydroxyC1-4alkyl, haloC1-4alkyl, dihaloC1-4alkyl, C1-4alkoxy, C1-4alkoxyC1-4alkyl, sulfanylC1-4alkyl, aminoC1-4alkyl, Nxe2x80x94(C1-4alkyl)aminoC1-4alkyl, N,N-di(C1-4alkyl)aminoC1-4alkyl or phenylC1-4alkyl; m is 0,1 or 2;
R12 and R13 are independently hydrogen or C1-4alkyl;
Ar1 is phenyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, thienyl, thiazolyl, furyl or oxazolyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3, wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; or Ar1 is pyrrolyl, pyrazolyl or imidazolyl, substituted by R2 and xe2x80x94(CH2)nR3 (the pyrrolyl, pyrazolyl or imidazolyl rings can bear a substituent on the sp3 hybridised ring nitrogen or Ar2 can be attached to Ar1(R12)R13CHxe2x95x90CHxe2x80x94 by the sp3 hybridised ring nitrogen);
R2 is a group of the Formula (2): 
wherein R7 is hydrogen or C1-4alkyl, R8 is xe2x80x94(CH2)qxe2x80x94R10 wherein q is 0-4 and R10 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy, C1-4alkoxy, carbamoyl, Nxe2x80x94C1-4alkyl carbamoyl, N,N-(diC1-4alkyl)carbamoyl, C1-4alkyl, phenyl, thienyl, or C1-4alkanoylamino, R9 is hydroxy, C1-6alkoxy, C3-9cycloalkyloxy, heterocyclyloxy, heterocyclylC1-4alkoxy or xe2x80x94NHxe2x80x94SO2xe2x80x94R11 wherein R11 represents, trifluoromethyl, C1-4alkyl, phenyl, heteroaryl, arylC1-4alkyl or heteroarylC1-4alkyl;
or R2 represents a lactone of Formula (3) 
xe2x80x83the group of Formula (2) or (3) having L or D configuration at the chiral alpha carbon in the corresponding free amino acid;
n is 0, 1, or 2;
R3 is phenyl or heteroaryl;
or R2 is a group of the Formula (4):
xe2x80x94CONHCH(R14)R15xe2x80x83xe2x80x83Formula (4)
xe2x80x83wherein R14 is xe2x80x94(CH2)qxe2x80x94R15 wherein q is 0-4 and R15 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy, C1-4alkoxy, carbamoyl, Nxe2x80x94C1-4alkyl carbamoyl, N,N-(diC1-4alkyl)carbamoyl, C1-4alkyl, phenyl, thienyl, or C1-4alkanoylamino; R14 is of the formula xe2x80x94CH2OR17 (wherein R17 is hydrogen, C1-4alkyl, phenyl, heteroaryl, C2-5alkanoyl, C1-4alkoxymethyl, phenoxymethyl or heteroaryloxymethyl), of the formula xe2x80x94COR18 or of the formula xe2x80x94CH2COR15 (wherein R18 is C1-4alkyl (optionally substituted by halo, cyano, C2-5alkanoyloxy, hydroxy, C1-4alkoxy or C1-4alkanoyl), phenyl, phenylC1-4alkyl, heteroaryl, heteroarylC1-4alkyl, C5-7cycloalkyl, C5-7cycloalkylC1-3alkyl, 2-(phenyl)ethenyl, 2-(heteroaryl)ethenyl or N-methoxy-N-methylamino); or R13 is morpholinoC1-4alkyl, pyrrolidin-1-ylC1-4alkyl or piperidin-1-ylC1-4alkyl wherein the morpholine, pyrrolidine and piperidine rings are optionally substituted by C1-4alkyl or C5-7cycloalkyl; or R13 is phenyl-1-hydroxyC1-4alkyl or heteroaryl-1-hydroxyC1-4alkyl;
phenyl and heteroaryl rings in R3, R5, R6, R11 and R15 (including R17 and R18) are independently optionally substituted on ring carbon atoms by up to three substituents selected from C1-4alkyl, halogen, hydroxy, C1-4alkoxy, C1-4alkoxycarbonyl, C1-4alkanoyl, C1-4alkanoyloxy, amino, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, nitro, cyano, carboxy, thiol, C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, C1-4alkanesulphonamido, Nxe2x80x94(C1-4alkylsulphonyl)xe2x80x94Nxe2x80x94C1-4alkylamino, aminosulfonyl, Nxe2x80x94(C1-4alkyl)aminosulfonyl, N,N-di(C1-4alkyl)aminosulfonyl, carbamoyl, Nxe2x80x94(C1-4alkyl)carbamoyl, N,N-(diC1-4alkyl)carbamoyl, carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl, N,N-(diC1-4alkyl)carbamoylC1-4alkyl, hydroxyC1-4alkyl and C1-4alkoxyC1-4alkyl and on ring NH groups (replacing hydrogen) by C1-4alkyl, C1-4alkanoyl, C1-4alkylsulfonyl, haloC1-4alkyl, difluoromethyl or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof
In this specification the generic term xe2x80x9calkylxe2x80x9d includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as xe2x80x9cisopropylxe2x80x9d are specific for the branched-chain version only. An analogous convention applies to other generic terms.
It is to be understood that, insofar as certain of the compounds of Formula (1) defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of inhibiting FTPase. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, inhibitory properties against FTPase may be evaluated using the standard laboratory techniques referred to hereinafter.
The term xe2x80x9cheterocyclylxe2x80x9d refers to a 5- or 6-membered monocyclic ring containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur.
The term xe2x80x9cheteroarylxe2x80x9d refers to a 5-10 membered monocyclic heteroaryl ring containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulphur.
The ring sp3 hybridised ring nitrogen in the pyrrolyl, pyrazolyl or imidazolyl rings is the ring nitrogen which can be substituted without becoming quaternised i.e. the ring  greater than NH nitrogen.
The term xe2x80x9chalogen xe2x80x9d refers to fluorine, chlorine, bromine and iodine. The term xe2x80x9ccarbamoylxe2x80x9d refers to xe2x80x94C(O)NH2. The term xe2x80x9cBOC xe2x80x9d refers to tert-butoxycarbonyl.
Examples of C1-4alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl and tert-butyl; examples of C1-4alkoxy include methoxy, ethoxy and propoxy; examples of C1-4alkanoyl include formyl, acetyl and propionyl; examples of C1-4alkanoyloxy include acetyloxy and propionyloxy; examples of C1-4alkylamino include methylamino, ethylamino, propylamino, isopropylamino, sec-butylamino and tert-butylamino; examples of di-(C1-4alkyl)amino include di-methylamino, di-ethylamino and N-ethyl-N-methylamino; examples of C1-4alkanoylamino include acetamido and propionylamino; examples of C1-4alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl; examples of C1-4alkylsulfanyl include methylsulfanyl, ethylsulfanyl, propylsulfanyl, isopropylsulfanyl, sec-butylsulfanyl and tert-butylsulfanyl; examples of C1-4alkylsulfinyl include methylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl, sec-butylsulfinyl and tert-butylsulfinyl; examples of C1-4alkylsulfonyl include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, sec-butylsulfonyl and tert-butylsulfonyl; examples of Nxe2x80x94(C1-4alkyl)carbamoyl include N-methylcarbamoyl and N-ethylcarbamoyl; examples of N,N-(diC1-4alkyl)carbamoyl include N,N-dimethylcarbamoyl and N-methyl-N-ethylcarbamoyl; examples of C1-4alkanesulfonamido include methanesulfonamido, ethanesulphonamido and propanesulfonamido; examples of C1-4alkylsulfonyl-Nxe2x80x94C1-4alkylamino include methylsulfonyl-N-methylamino, ethylsulfonyl-N-methylamino and propylsulfonyl-N-methylamino; examples of fluoroC1-4alkyl include fluoromethyl, 2-fluoroethyl and 3-fluoropropyl; examples of difluoroC1-4alkyl include difluoromethyl, 2,2-difluoroethyl and 3,3-difluoropropyl; examples of carbamoylC1-4alkyl include carbamoylmethyl, carbamoylethyl and carbamoylpropyl; examples of Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl include N-methyl-carbamoylmethyl and N-ethyl-carbamoylethyl; examples of N,N-(diC1-4alkyl)carbamoylC1-4alkyl include N,N-dimethylcarbamoylethyl and N-methyl-N-ethylcarbamoylethyl; examples of hydroxyC1-4alkyl include hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-hydroxypropyl, 2-(hydroxymethyl)propyl and hydroxybutyl; examples of C1-4alkoxyC1-4alkyl include methoxyethyl, ethoxyethyl and methoxybutyl; examples of sulfanylC1-4alkyl include sulfanylmethyl, sulfanylethyl, sulfanylpropyl; and examples of Nxe2x80x94(C1-4alkyl)aminoC1-4alkyl include N-methyl-aminomethyl and N-ethyl-aminoethyl.
Examples of 5- or 6-membered heteroaryl ring systems include imidazole, triazole, pyrazine, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole and thiophene.
Preferably the NH group in imidazole is unsubstituted or substituted by C1-4alkyl. Examples of heterocyclyl rings include pyrrolidinyl, morpholinyl, piperidinyl, dihydropyridinyl and dihydropyrimidinyl.
Preferred heteroatoms are N and S, especially N. In general, attachment of heterocyclic rings to other groups is via carbon atoms.
Examples of values for R8 in Formula (2) are side chains of lipophilic amino acids including such as for example methionine, phenylglycine, phenylalanine, serine, leucine, isoleucine or valine. The L configuration in the corresponding free amino acid is preferred. Examples of amino acid side chains are set out below.
The lactone in Formula (3) can be formed from a group of Formula (2) when R9 is OH to give a carboxyl and R8 is xe2x80x94CH2xe2x80x94CH2xe2x80x94OH where R8 and R9 together lose a water molecule to form part of a dihydrofuran-2-one heterocyclic ring.
Preferably, phenyl and heteroaryl rings in R3, R5, R6, R11 and R15 (including R17 and R18) are independently optionally substituted on ring carbon atoms by up to two substituents selected from C1-4alkyl, halogen, hydroxy, C1-4alkoxy, C1-4alkoxycarbonyl, C1-4alkanoyl, C1-4alkanoyloxy, amino, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, cyano, carboxy, C1-4alkylsufanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, C1-4alkanesulphonamido, carbamoyl, Nxe2x80x94(C1-4alkyl)carbomoyl, N,N-(diC1-4alkyl)carbamoyl and on ring NH groups (replacing hydrogen) by C1-4alkyl or C1-4alkanoyl.
Preferably R12 and R13 are independently hydrogen or methyl.
Most preferably R12 and R13 are hydrogen. Preferably R6 is hydrogen, C1-4alkyl, hydroxyC1-4alkyl, aminoC1-4alkyl, fluoroC1-4alkyl, difluoroC1-4alkyl, C1-4alkoxy or C1-4alkoxyC1-4alkyl.
More preferably R6 is hydrogen, methyl, fluoromethyl, difluoromethyl, methoxy or methoxymethyl.
Most preferably R6 is hydrogen or methyl.
Preferably m is 0 or 1.
Preferably R5 is hydrogen or methyl. More preferably R5 is hydrogen.
In a particular aspect Ar1 is 1 -methylimidazol-5-yl.
Preferably Ar2 is phenyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, thienyl, thiazolyl, or oxazolyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3. Most preferred is where Ar2 is phenyl, pyridinyl or thienyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(H2)nR3.
More preferably Ar2 is phenyl, pyridinyl or thienyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Yet more preferably Ar2 is phenyl or pyridyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Most preferably Ar2 is phenyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Preferably, when n is 0, Ar2 is substituted by R2 in the 4-position and xe2x80x94(CH2)nR3 in the 3- or 5-position and when n is 1 or 2, Ar2 is substituted by R2 in the 3- or 5-position and xe2x80x94(CH2)nR2 in the 4-position. The positions indicated are relative to the point of attachment of Ar2 to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94.
Preferably n is 0 or 2. In a particular aspect n is 0.
R2 is preferably a group of formula: 
R7 is preferably hydrogen or methyl, especially hydrogen. In R8, q is preferably 1-4, more preferably 1 or 2, especially 2.
Within R8, R10 is preferably C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy. More preferably R10 is methylsulfanyl or methylsulfonyl.
R9 is preferably hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocyclyloxy or heterocyclylC1-4alkoxy. More preferably R9 is hydroxy, methoxy, propoxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin-4-yloxy or morpholinoC1-4alkyl. Most preferably, R9 is methoxy, prop-2-oxy, n-butoxy, tert-butoxy or cyclopentyloxy.
Preferably R11 in R9 is phenyl.
Preferred substituents for NH groups in heterocyclic groups in R9 include methyl, ethyl, acetyl, propionyl, fluoromethyl, difluoromethyl and trifluoromethyl.
More preferred substituents for NH groups in heterocyclic groups in R9 include methyl and acetyl.
Preferred substituents for ring carbon atoms in phenyl or heteroaryl groups in R11 include methyl, halo, C1-4alkanoyl, nitro, cyano, C1-4alkylsulfinyl, C1-4alkylsulfonyl, carbamoyl, C1-4alkylcarbamoyl and diC1-4alkylcarbamoyl.
Formula (4) is preferably a group of formula: 
In R14, q is preferably 1-4, more preferably 1 or 2, especially 2.
Within R14, R14 is preferably C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy. More preferably R16 in R14 is methylsulfanyl or methylsulfonyl. Preferably R17 in R15 is hydrogen or phenyl.
Most preferably R17 in R15 is hydrogen.
Preferably R18 in R15 is C1-4alkyl, phenyl, phenylC1-3alkyl, heteroaryl, heteroarylC1-3alkyl or C5-7cycloalkylC1-3alkyl.
More preferably R18 in R15 is C1-4alkyl, phenyl, phenylC1-3alkyl or heteroaryl.
Most preferably R18 in R15 is C1-4alkyl, phenyl or benzyl.
Preferably when R18 is C1-4alkyl, it is optionally substituted by halo, cyano or C2-6alkanoyloxy.
Preferably morpholinoC1-4alkyl is morpholinomethyl, pyrrolidin-1-ylC1-4alkyl is pyrrolidin-1-ylmethyl and piperidin-1-ylC1-4alkyl is piperidin-1-ylmethyl.
In one aspect R15 is morpholinomethyl.
More preferably R15 is hydroxymethyl, benzylcarbonyl, 3-(pyridyl)propionyl or morpholinomethyl.
Most preferably R15 is hydroxymethyl or benzylcarbonyl. R14 is xe2x80x94(CH2)qR16 wherein q is 0-4 and R16 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy; or R15 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl; or R15 is of the formula xe2x80x94CH2OR17 wherein R17 is hydrogen or phenyl; or R15 is of the formula xe2x80x94COR18 or xe2x80x94CH2COR18 wherein R18 is C1-4alkyl, phenyl, phenylC1-3alkyl, heteroaryl, heteroarylC1-3alkyl or C5-7cycloalkylC1-3alkyl; Preferably R3 is phenyl, pyridyl or thiazolyl.
Most preferably R3 is phenyl.
Preferred substituents for ring carbon atoms in R3 include C1-4alkyl, halo, trifluoromethyl, C1-4alkoxy, nitro, cyano and C1-4alkoxy C1-4alkyl.
More preferred substituents for ring carbon atoms in R3 include methyl, fluoro, chloro, trifluoromethyl, methoxy, nitro, cyano and methoxymethyl.
When R3 is phenyl it is preferably mono-substituted by fluoro, chloro or cyano or di-substituted by fluoro and triflouromethyl, chloro and trifluoromethyl, fluoro and fluoro or chloro and chloro.
A preferred substituent for a ring NH group in a heteroaryl group in R3 is C1-4alkyl, particularly methyl.
When R3 is phenyl it is preferably substituted in the 4-position, and when di-substituted it is preferably substituted in the 2- and 4-positions.
Preferably n is 0 or 2.
Preferably R7 is hydrogen.
Preferably R8 is xe2x80x94(CH2)xe2x80x94R10 is C1-4alkylsulfonyl or C1-4alkylsulfonyl.
Preferably R9 is hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocyclyloxy or heterocyclylC1-4alkoxy.
A preferred compound of the invention is a compound of the Formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen, C1-4alkyl, fluoroC1-4alkyl, difluoroC1-4alkyl, C1-4alkoxy or C1-4alkoxyC1-4alkyl;
m is 0 or 1;
R12 and R13 are independently hydrogen or methyl;
Ar2 is phenyl or pyridyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR10 and wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; and
n is 0, 1 or 2;
R2 is of the formula (2) wherein R7 is hydrogen or methyl;
R5 is xe2x80x94(CH2)qR10 wherein q is 0-4 and R10 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy;
R9 is hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocycloxy or heterocyclylC1-4alkoxy;
or R2 is of the formula (3);
R3 is phenyl, pyridyl or thiazolyl; and phenyl, heteroaryl and heterocyclyl rings in R3 and R9 are independently optionally substituted on ring carbon atoms by one or two substituents selected from C1-4alkyl, halo, C1-4alkoxy, C1-4alkanoyl, nitro, cyano, C1-4alkylsulfinyl, C1-4alkylsulfonyl, carbamoyl, C1-4alkylcarbamoyl and diC1-4alkylcarbamoyl; and optionally substituted on ring NH groups by C1-4alkyl, C1-4alkanoyl, fluoromethyl, difluoromethyl or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
A more preferred compound of the invention is a compound of the formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen, methyl, fluoromethyl, difluoromethyl, methoxy or methoxymethyl;
m is 0 or 1;
R12 and R13 are independently hydrogen or methyl;
Ar2 is phenyl or pyridyl; the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3 and wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; and n is 0, 1 or 2;
R2 is of formula (2) wherein R7 is hydrogen or methyl;
R8 is (CH2)qR10 wherein q is 1 or 2, and
R10 is methylsulfanyl or methylsulfonyl;
R9 is hydroxy, methoxy, prop-2-oxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin -4 -yloxy, or morpholinoC1-4alkyl; or R2 is of the formula (3);
R3 is phenyl optionally substituted by one or two substituents selected from C1-4alkyl, halo, C1-4alkoxy, nitro, cyano, C1-4alkoxyC1-4alkyl and trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
An even more preferred compound of the invention is a compound of the formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen or methyl;
m is 0 or 1;
R11 and R12 are hydrogen;
Ar2 is phenyl; the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3 and wherein
Ar2 is attached to Ar1(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom;
and n is 0, 1 or 2;
R2 is of the Formula (2) wherein R7 is hydrogen;
R8 is xe2x80x94(CH2)8R10 wherein q is 2 and within Formula (2)
R10 is methylsulfanyl or methylsulfonyl;
R9 is hydroxy, methoxy, prop-2-oxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin-4-yloxy, or 2-morpholinoprop-2-yl; or within Formula (4)
R15 is of the fomula xe2x80x94CH2OR17 wherein R17 is hydrogen; or R15 is of the formula xe2x80x94COR18 or xe2x80x94CH2COR15 wherein R18 is C1-4alkyl, phenyl, phenylC1-3alkyl or heteroaryl; or
R15 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl;
R3 is phenyl optionally substituted by fluoro, chloro, cyano or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
Preferably Ar1C(R12)R13xe2x80x94 and Ar2 are on opposite sides of the double bond (this gives the E isomeric configuration).
Particular compounds of the present invention include those compounds specifically described in the Examples; or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
Compounds of Formula (1) may form salts which are within the ambit of the invention. Pharmaceutically acceptable salts are preferred although other salts may be useful in, for example, isolating or purifying compounds.
When the compound contains a basic moiety it may form pharmaceutically-acceptable salts with a variety of inorganic or organic acids, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric or maleic acid. A suitable pharmaceutically-acceptable salt of the invention when the compound contains an acidic moiety is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
Solvates, for example hydrates, are also within the ambit of the invention and may be prepared by generally known methods.
Various forms of prodrugs are well known in the art. For examples of such prodrug derivatives, see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 xe2x80x9cDesign and Application of Prodrugsxe2x80x9d, by H. Bundgaard p. 113-191 (1991);
c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and
e) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984).
Examples of pro-drugs include in vivo hydrolysable esters of a compound of the Formula I. Suitable pharmaceutically-acceptable esters for carboxy include C1-4alkyl esters, C5-8cycloalkyl esters, cyclic amine esters, C1-6alkoxymethyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters for example 5-methyl-1,3-dioxolen-2-onylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl wherein alkyl, cycloalkyl and cyclicamino groups are optionally substituted by, for example, phenyl, heterocyclcyl, alkyl, amino, alkylamino, dialkylamino, hydroxy, alkoxy, aryloxy or benzyloxy, and may be formed at any carboxy group in the compounds of this invention.
According to another aspect of the invention there is provided a pharmaceutical composition comprising a compound as defined in Formula (1) or an individual compound listed above together with a pharmaceutically-acceptable diluent or carrier. A preferred pharmaceutical composition is in the form of a tablet.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30xcexc or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula (1) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula (1) are useful in treating diseases or medical conditions which are due alone or in part to the effects of farnesylation of ras.
In using a compound of the Formula (1) for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred.
Compounds of this invention may be useful in combination with known anti-cancer and cytotoxic agents. If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate. According to another aspect of the invention there is provided a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, for use as a medicament.
According to another aspect of the invention there is provided a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, for use in preparation of a medicament for treatment of a disease mediated through farnesylation of ras.
According to another aspect of the present invention there is provided a method of treating ras mediated diseases, especially cancer, by administering an effective amount of a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, to a mammal in need of such treatment.
Diseases or medical conditions may be mediated alone or in part by farnesylated ras. A particular disease of interest is cancer. Specific cancers of interest include:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;
hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma and Burketts lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and
other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma.
The compounds of Formula (1) are especially useful in treatment of tumors having a high incidence of ras mutation, such as colon, lung, and pancreatic tumors. By the administration of a composition having one (or a combination) of the compounds of this invention, development of tumors in a mammalian host is reduced.
Compounds of Formula (1) may also be useful in the treatment of diseases other than cancer that may be associated with signal transduction pathways operating through Ras, e.g., neuro-fibromatosis.
Compounds of Formula (1) may also be useful in the treatment of diseases associated with CAAX-containing proteins other than Ras (e.g., nuclear lamins and transducin) that are also post-translationally modified by the enzyme farnesyl protein transferase.
Although the compounds of the Formula (1) are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit the effects of activation of ras by farnesylation. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
In another aspect the present invention provides a process for preparing a compound of the Formula (1) or a pharmaceutically-acceptable salt prodrug or solvate thereof which process comprises:
deprotecting a compound of the formula (5) 
wherein Ar1xe2x80x2 is Ar1 or protected Ar1 and Ar2xe2x80x2 is Ar2 or protected Ar2 and R12 and R13 are as hereinabove defined; wherein at least one protecting group is present; and thereafter if necessary:
(i) forming a pharmaceutically-acceptable salt,
(ii) forming a prodrug,
(iii) forming a solvate.
Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxy protecting group may be the residue of an ester-forming aliphatic or araliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain C1-12alkyl groups (for example isopropyl, t-butyl); lower alkoxy lower alkyl groups (for example methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (for example acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (for example 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl); phenyl lower alkyl groups (for example benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (for example trimethylsilyl and t-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (for example trimethylsilylethyl); and C1-6alkenyl groups (for example allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxy protecting groups include for example acid-, base-, metal- or enzymically-catalysed hydrolysis.
Examples of hydroxy protecting groups include lower alkyl groups (for example t-butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example t-butoxycarbonyl); lower alkenyloxycarbonyl groups (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (for example trimethylsilyl, t-butyldimethylsilyl) and phenyl lower alkyl (for example benzyl) groups.
Examples of amino protecting groups include formyl, aralkyl groups (for example benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (for example t-butoxycarbonyl); lower alkenyloxycarbonyl (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (for example trimethylsilyl and t-butyldimethylsilyl); alkylidene (for example methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.
The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley and Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis, 2nd Edition, by Green et al., published by John Wiley and Sons for general guidance on protecting groups.
Compounds of the formula (1) and (5) can be formed by:
(i) reacting a compound of the formula (6) with a compound of the formula (7): 
or (ii) converting one value of R9 in R2 into another value of R9;
or (iii), where the compound of formula (I) includes alternatives of formula (2) and (3), reacting a compound in which R2 in Ar2xe2x80x2 is carboxy with a compound of the formula (8): 
or (iv), where the compound of formula (I) includes alternative of formula (4), reacting a compound in which R2 in Ar2 is carboxy with a compound of formula (8a).
or (v) when Ar1xe2x80x2 is of the formula (B) or (C) (optionally protected), reacting a compound of the formula (9) or (9a) with a compound of the formula (10): 
xe2x80x83wherein p, Ar1xe2x80x2, Ar2xe2x80x2, Ar3xe2x80x2 R7 and R8 are as hereinabove defined, R21 is R9 or protected R9, L1 and L2 are leaving groups and Pxe2x80x2 is an amino-protecting group or R5 providing it is not hydrogen; and thereafter if necessary:
(i) removing any protecting groups;
(ii) forming a pharmaceutically-acceptable salt, prodrug or solvate thereof.
The reaction between compounds of the formula (6) and (7) is conveniently carried out under conditions know for the Wittig reaction.
Suitable Wittig reaction conditions include using a polar aprotic organic solvent such as THF or methylene chloride in the presence of a crown ether and an alkali metal carbonate, such as potassium carbonate, preferably at xe2x88x9210xc2x0 C. to ambient temperature. C18 HPLC may be used to separate E and Z isomers if desired.
The Wittig reaction can be used to prepare a compound wherein R2 in Ar2xe2x80x2 is an alkoxycarbonyl group which may be hydrolysed to a carboxy group. If Ar1xe2x80x2 is of the formula (A), and the latter route is chosen and basic conditions are used, the E and Z isomers produced by the Wittig reaction are generally not separated if the E isomer is wanted, because the base-hydrolysis step causes isomerisation of the Z isomer to the E isomer.
A compound of the formula (6) is generally prepared by reducing the corresponding ester to the aldehyde. A suitable reducing agent is for example DIBAL which is used in an inert non-polar solvent such as diethyl ether at low temperature, for example in the range xe2x88x9278xc2x0 C. to 0xc2x0 C. Other suitable reducing agents are known in the art.
Alternatively, the ester could be reduced to the corresponding alcohol with a reducing agent such as lithium aluminium hydride or sodium borohydride and the alcohol oxidised to the aldehyde with an oxidising agent such as pyridinium chlorochromate.
The ester used to form a compound of the formula (6) can be prepared by introducing Are into a compound of the formula L4CH2COOR, wherein L4 is leaving group such as bromo, in the presence of a base such as sodium hydride, sodium hydroxide or potassium carbonate.
A compound of the formula (7) may be known in the art (for example see International Patent Application publication no. WO 98/32741) or may be prepared by reacting a compound of the formula Ar2xe2x80x2CH2Br with triphenylphosphine.
A compound of the formula Ar2xe2x80x2CH2Br may be formed by brominating a compound of the formula Ar2xe2x80x2CH3 with a suitable brominating agent such as N-bromosuccinimide.
A compound of the formula Ar2xe2x80x2CH3 wherein the Ar2xe2x80x2 group is substituted by R2 and xe2x80x94(CH2)nR3 group may be prepared from a compound of the formula Ar2xe2x80x2CH3 wherein the Ar2 group is substituted by xe2x80x94(CH2)nR3 and xe2x80x94COOHxe2x80x2 using methods described above for the conversion of xe2x80x94COOH to R2. This latter Ar2xe2x80x2CH3 compound could be prepared from a compound of the formula Ar2xe2x80x2CH3 wherein the Ar2xe2x80x2 group is substituted by a protected carboxy group (xe2x80x94COOP2) and a leaving group (L3). When n is 0, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound is conveniently reacted with aryl (or heteroaryl) boronic acid in the presence of a palladium catalyst such as palladium tetrakis (triphenylphosphine) palladium (0) under conditions known for the Suzuki reaction (Synth. Commun. 11, 513 (1981)). An aprotic organic solvent such as dimethyl ether (DME), toluene, dimethylsulphoxide (DMSO) or THF is generally used and a base such as sodium bicarbonate, sodium carbonate and sometimes sodium hydroxide. A fluoride such as caesium fluoride could be used instead of the base (J. Org. Chem. 1994, 59, 6095-6097). Preferably L3 is bromo or triflate.
When n is 1, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound wherein L3 is bromo or chloro, is conveniently reacted with a benzyl (or heteroarylmethyl) zinc chloride or a benzyl (or heteroarylmethyl)-magnesium bromide in the presence of a nickel or palladium catalyst, such as bis(triphenylphosphine)palladium (II) chloride or Pd2(dba)3, in an inert organic solvent such as tetrahydrofuran (THF). For example see the conditions used for the xe2x80x98Negishixe2x80x99 reaction (J. Org. Chem. 42 (10), 1821-1822, 1977).
When n is 2, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound is conveniently reacted with a styrene under conditions known for the Heck reaction. Briefly this involves an inorganic or organic base such as triethylamine, a palladium catalyst such as bis (o-tolylphosphine)palladium (II) chloride in water. (Acc. Chem. Res. 12, 146-151 (1979), J. Organometallic Chem. 486, 259-262 (1995)).
The resulting alkene can then be reduced using standard methods known in the art, for example, catalytic hydrogenation.
Alternatively the alkyne could be formed by reacting a CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound, wherein L3 is triflate or bromo, with a phenyl acetylene in the presence of an organic base such as triethylamine and a palladium catalyst such as palladium tetrakis (triphenylphosphine). For example see the conditions used for the Sonogashira reaction (J. Org. Chem. 1993,58, 6614-6619).
The resultant alkyne can be reduced using standard methods known in the art, for example, catalytic hydrogenation.
The carboxy-protecting group may then be removed.
A compound of the formula (1) in which R9 in R2 is alkoxy can conveniently be hydrolysed to another compound of the formula (1) in which R9 is hydroxy using standard methods known in the art. For example, the alkoxy group could be subjected to acid or base hydrolysis with, for example, in the case of base hydrolysis, aqueous sodium hydroxide solution in an organic solvent such as an alcohol in a temperature range of ambient temperature to 60xc2x0 C. When R9 is a hydroxy group the carboxy group in a compound of the formula (1) can be converted to an acylsulphonamide by reacting the carboxy group with the appropriate sulphonamido group in the presence of an organic base such as triethylamine or dimethylaminopyridine, in an inert organic solvent such as dimethylformamide (DMF), in temperature range of xe2x88x9220xc2x0 C. to ambient temperature.
The reaction between a compound in which R2 in Ar2xe2x80x2 is carboxy and a compound of the formula (8) and (8a) is generally carried out in the presence of a reagent that converts the carboxy group into a reactive ester, for example a carbodiimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) or pentafluorophenyl, and in the presence of an organic base such as N-methylmorpholine. The reaction is usually carried out in the temperature range of xe2x88x9220xc2x0 C. to ambient temperature. The reagents, 1-hydroxybenzotriazole and dimethylaminopyridine (DMAP), are often added to assist the reaction (see Chem. Ber. 103, 788, 2024 (1970), J. Am. Chem. Soc. 93, 6318 (1971), Helv. Chim. Acta. 56, 717, (1973)). Suitable solvents include DMF and dichloromethane.
For examples of suitable conditions for amide bond forming reactions see International Patent Application No. WO 98/07692.
A compound of the formula (1) in which R2 in Ar2xe2x80x2 is carboxy can be prepared by reacting a compound of the formula (6) with a compound of the formula (7), or a compound of the formula (9) or (9a) with a compound of the formula (10) wherein R2 in Ar2xe2x80x2 is protected carboxy and subsequently removing the carboxy-protecting group.
A compound of the formula (8a) wherein R22 is of the formula xe2x80x94COR18 can be formed via the intermediate NH2CH(R21)CON(OMe)Me which itself is formed by reacting together NH2CH(R21)COOH with N,O-dimethylhydroxylamine under standard amide bond forming conditions. A compound of the formula NH2CH(R21)CON(OMe)Me is then conveniently reacted with a Gringard reagent (such as PhCH2MgCl) to form a compound of the formula (8).
Alternatively, when R18 contains an alkyl chain linked to the carbonyl group, a compound of the formula NH2CH(R21)CON(OMe)Me can be converted to the corresponding dimethylphosphono compound (NH2CH(R21)COP(O)(OMe)2) by reacting the former compound with dimethylmethylphosphonate in the presence of a strong base such as n-butyl lithium. A compound of the formula (8) can be formed by reacting the dimethylphosphono compound with the appropriate aldehyde or ketone under conditions known for the Wittig or Emmons-Horner reactions.
A compound of the formula (8) wherein R22 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl is conveniently prepared by reacting NH2CH(R21)COOH with the appropriate heterocyclic ring under standard amide bond forming conditions to form a compound of the formula (8), wherein R15 is heterocyclylcarbonyl, and subsequently reducing the carbonyl group to a methyl group with a reducing agent such as lithium aluminium hydride.
A compound of the formula NH2CH(R21)COOH can be extended by one carbon length to produce a compound of the formula NH2CH(R21)CH2COOH using the Arndt-Eistert homologation method. For example by converting the carboxy group to an acid chloride, converting the latter to the diazoketone and hydrolysing this to the carboxylic acid. This homologation method could be used to produce subsequent homologues. A compound of the formula NH2CH(R21)CH2COOH and homologues may be used to prepare a compound of the formula NH2CH(R21)R22 wherein R22 is of the formula xe2x80x94CH2COR15, morpholino C1-4alkyl, pyrrolidin-1-ylC1-4alkyl or piperidin-1-ylC1-4alkyl.
An anion of the formula (9) is conveniently formed with a strong base, such as n-butyl lithium. This generally forms an anion at the 2-position of the imidazole compound. The anion formation is generally carried out at low temperature e.g. xe2x88x9278xc2x0 C., in an inert or organic solvent such as diethyl ether.
The anion (usually the lithium anion) of a compound of the formula (9) is normally converted to an organozinc reagent in the presence of lithium chloride, zinc iodide and copper (I) cyanide and this complex is reacted with a compound of the formula (10) in an inert organic solvent such as tetrahydrofuran in a temperature range of xe2x88x9278xc2x0 C. to ambient temperature (see the review of organozinc reagents by Paul Knochel and Robert D Singer in Chem. Rev. 1993, 93, 2117-2188).
The anion of a compound of the formula (9a) is conveniently formed as a Grignard reagent by reacting the latter compound with an alkyl magnesium bromide in an ether at low temperature. The resulting Grignard reagent is typically reacted with a compound of the formula (10) in the presence of copper (I) cyanide and lithium chloride, allowing the temperature to rise from xe2x88x9278xc2x0 C. to ambient temperature. Preferably L1 in the compound of the formula (9a) is halo, for example iodo or bromo.
Preferably L2 in the compound of the formula (10) is halo, for example bromo.
A compound of the formula (10) may be prepared by brominating the corresponding hydroxy compound with a brominating agent such as tetrabromomethane/triphenylphosphine in a suitable solvent such as dichloromethane. The hydroxy compound is typically prepared by reacting a compound of the formula (7) and glycoaldehyde under conditions known for the Wittig reaction such as those described above for the reaction between compounds of the formulae (6) and (7).
The order in which the various groups are introduced can be varied depending upon the nature of the desired final product. In general, the preferred last step is the formation or modification of R2 in Ar2xe2x80x2. For example, when R9 is hydroxy, the hydrolysis of the compound wherein R9 is alkoxy, or when R9 is other than hydroxy, the conversion of a carboxy group to R2 using the amide bond-forming conditions described above.
When Ar1xe2x80x2 of the formula (A), it is preferable to form the alkene which contains a protected carboxy group later to be converted to R2, by reacting together compounds of the formulae (6) and (7).
When Ar2xe2x80x2 is of the formula (B) or (C) the alkene which contains a protected carboxy group later to be converted to R2, is preferably formed by reacting together a compound of the formula Ar2xe2x80x2CH2P+Ph3Br (wherein Ar2xe2x80x2 is substituted by xe2x80x94COOP2 in place of R2) and glycoaldehyde, converting the resultant hydroxy compound to a compound of the formula (9) (wherein Ar2xe2x80x2 is substituted by xe2x80x94COOP2 in place of R2) and reacting the latter compound with a compound of the formula (8) or (8a).
Optionally substituents in a compound of the formula (1) and (5) or intermediates in their preparation may be converted into other optional substituents. For example an alkylthio group may be oxidised to an alkylsulphinyl or alkysulphonyl group, a nitro group reduced to an amino group, a hydroxy group alkylated to a methoxy group, or a bromo group converted to an alkylthio group.
Various substituents may be introduced into compounds of the formulae (1) and (5) and intermediates in this preparation, when appropriate, using standard methods known in the art. For example, an acyl group or alkyl group may be introduced into an activated benzene ring using Friedel-Crafts reactions, a formyl group by formylation with titanium tetrachloride and dichloromethyl ethyl ester, a nitro group by nitration with concentrated nitric acid concentrated sulphuric acid and bromination with bromine or tetra(n-butyl)ammonium tribromide.
It will be appreciated that, in certain steps in the reaction sequence to compounds of the formula (1), it will be necessary to protect certain functional groups in intermediates in order to prevent side reactions. Deprotection may be carried out at a convenient stage in the reaction sequence once protection is no longer required.
Biological activity was tested as follows:
(i) In-vitro Assay
The following stock solutions were used and the assays were conducted in 96 well plates: TRIS Buffer (500 mM TRIS, 5 mM MgCl2.6H2O, pH=8.0); Farnesyl pyrophosphate (6.4 mg/ml); Aprotinin (1.9 mg/ml); Ki-ras (0.5 mg/ml, stored at xe2x88x9280xc2x0 C.); Acid ethanol (850 ml absolute ethanol+150 ml concentrated HCl).
Farnesyl protein transferase (FPT) was partially purified from human placenta by ammonium sulphate fractionation followed by a single Q-Sepharose(trademark) (Pharmacia, Inc) anion exchange chromatography essentially as described by Ray and Lopez-Belmonte (Ray K P and Lopez-Belmonte J (1992) Biochemical Society Transactions 20 494-497). The substrate for FPT was Kras (CVIM C-terminal sequence). The cDNA for oncogenic val 12 variant of human c-Ki-ras-2 4B was obtained from the plasmid pSW11-1 (ATCC). This was then subcloned into the polylinker of a suitable expression vector e.g. pIC147. The Kras was obtained after expression in the E. coli strain, BL21. The expression and purification of c-KI-ras-2 4B and the val12 variant in E. coli has also been reported by Lowe et al (Lowe P N et al. J. Biol. Chem. (1991) 266 1672-1678). The farnesyl protein transferase enzyme preparation was stored at xe2x88x9280xc2x0 C.
The farnesyl transferase solution for the assay contained the following: dithiothreitol (DTT)(0.6 ml of 7.7 mg/ml), TRIS buffer (0.6 ml), aprotinin (0.48 ml), distilled water (1.2 ml), farnesyl transferase (0.6 ml of the crude enzyme preparation prepared as described above), zinc chloride (12 xcexcl of 5 mM). This was left at ambienttemperature for 30 minutes. After this incubation 60 xcexcl Ki-ras solution was added and the whole left to incubate for a further 60 minutes prior to use in the assay.
Assays were performed in 96 well plates as follows: 10 xcexcl of test compound solution was added to each well. Then 30 xcexcl farnesyl transferase solution (above) was added and the reaction started by addition of 10 xcexcl radiolabelled farnesyl pyrophosphate solution. After 20 minutes at 37xc2x0 C. the reaction was stopped with 100 xcexcl acid ethanol (as described in Pompliano D L et al (1992) 31 3800-3807). The plate was then kept for 1 hour at 4xc2x0 C. Precipitated protein was then collected onto glass fibre filter mats (B) using a Tomtec(trademark) cell harvester and tritiated label was measured in a Wallac(trademark) 1204 Betaplate scintillation counter. Test compounds were added at appropriate concentrations in DMSO (3% final concentration in test and vehicle control).
(ii) Intracellular Farnesylation Assay
HER313A cells (Grand et al, 1987 Oncogene 3, 305-314) were routinely cultured in Dulbecos Modified Essential Medium (DMEM) plus 10% foetal calf serum (FCS). For the assay HER313A cells were seeded at 200,000 cells/well in a volume of 2.5 ml in a 6 well tissue culture plate. After an overnight incubation at 37xc2x0 C. in 10% CO2 the medium was removed and replaced with methionine-free minimal essential medium (MEM) and the cells incubated as above for 2 hours. After this time the medium was removed and replaced by methionine-free MEM (1 ml) and test compound (1-3 xcexcl). The plates were then incubated for a further 2 hours as described above and then 30 xcexcCi of 35S-methionine added to each well. The plate was then incubated overnight as described above. The medium was then removed and the cells were lysed with lysis buffer (1 ml) (composed of 1000 ml phosphate buffered saline, 10 ml trition X-100, 5 g sodium deoxycholate, 1 g sodium dodecylsulphate) containing aprotinin (10 xcexcl/ml), the plate scrapped and then left for 10 minutes at 4xc2x0 C. The lysate was then clarified by centrifugation. To 0.8 ml of the clarified lysate 80 xcexcl of Y13-259 pan-Ras antibody (isolated from the hybridomaxe2x80x94American Tissue Culture Collection Accession Number CRL-1742) (final concentration approximately 1 xcexc/ml, the exact working concentration was optimised for each batch of antibody isolated) and protein G beads (30 xcexcof 0.5 xcexcg/ml) were added and the mixture incubated overnight with constant agitation. The pellet was then collected by centrifugation, washed and separated by SDS PAGE using a 15% gel. Radioactive bands were detected using a phosphorimager system.
(iii) Morphology and Proliferation Assay
MIA PaCa 2 cells (American Tissue Culture Collection Accession Number: CRL-1420) were routinely cultured in Dulbecos Modified Essential Medium (DMEM) plus 10% FCS in a 162 cm2 tissue culture flask. For the assay the cells were seeded at 16,000 cells/well, in 12 well plates, in DMEM containing 5% charcoal dextran treated stripped FCS (1 ml) (obtained from Pierce and Warriner). The cells were then incubated overnight at 37xc2x0 C. in 10% CO2. Test compound was then added (10 xcexcl) and the cells incubated for 6 days as described above. On days 1, 2, 3 and 6 the cells were monitored for signs of morphological change and toxicity. On day 6 the cells were removed from the plate using trypsin/EDTA and counted to determine the proliferation rate.
Although the pharmacological properties of the compounds of the Formula (1) vary with structural change as expected, in general compounds of the Formula (1) possess an IC50 in test (i) above in the range, for example, 0.00005 to 50 xcexcM in test (i). Thus by way of example the compound of Example 29 herein has an IC50 of approximately 0.0004 xcexcM in test unacceptable toxicity was observed at the effective dose for compounds tested of the present invention.